The overall goal of this proposal is to understand the physiological function of Ral proteins. They represent a distinct evolutionary branch of the ras gene family. Their encoded proteins bind and hydrolyze GTP and have recently been found to be localized primarily to non-Golgi clathrin coated vesicles. Moreover, a Ral-specific GTPase activating protein (Ral-GAP) has also been detected in clatherin coated vesicles, suggesting that Ral proteins have a regulatory role in receptor-mediated endocytosis. Since this process is involved in the metabolism of cell surface receptors, Ral proteins likely influence the process of signal transduction emanating from the plasma membrane. Genetic and biochemical studies based on our present knowledge of GTP binding proteins and clathrin coated vesicle function will be integrated to pursue this goal. One approach will be to characterize proteins that interact with Ral, such as Ral-GAP. In addition, an attempt will be made to generate dominant inhibitory Ral mutants and neutralizing antibodies that will be used to study the consequence of inhibiting endogenous Ral protein function in mammalian cells. Finally, changes in Ral-GAP activity and in the proportion of Ral in the active GTP state in cells will be measured in response to specific signal transduction signals and alterations in receptor-mediated endocytosis.